Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease

Defects in the PKD1 and PKD2 genes cause autosomal dominant polycystic kidney disease (PKD) in ~1 in 1000 adults worldwide. These genes encode polycystin-1 and polycystin-2, which are membrane proteins thought to be involved in a calcium signal transduction cascade that controls epithelial proliferation and differentiation. Individuals with mutations in these genes develop cysts in the ducts and tubules of their kidneys. These cysts lead to kidney failure in ~50% of affected individuals [1]. Similar to autosomal dominant PKD, autosomal recessive PKD also results in the formation of kidney cysts, but most human cases are caused by defects in a gene of unknown function [2]. How dominant and recessive PKD are related is unclear, but three recent findings suggest that both forms of the disease involve the non-motile primary cilia that extend from the kidney epithelial cells into the lumen of the collecting ducts and tubules. First, the Caenorhabditis elegans homologues of polycystin-1 and polycystin-2 are localized to the nematode's sensory cilia [3]. Second, the defective gene in the Tg737 orpk [4] recessive PKD mouse encodes a protein, IFT88, involved in intraflagellar transport and necessary for assembly of kidney primary cilia [5]. Finally, the defective gene in the cpk recessive PKD mouse encodes a novel ciliary protein [6]. Based on these findings, we hypothesize that the polycystins are located on primary cilia, and that the primary cilia have a sensory role in the polycystin-signaling pathway. To determine if polycystin-2 is localized to the primary cilium, we examined its distribution in ciliated cultures of human and mouse kidney cells by immuno-fluorescence microscopy using the extensively characterized YCC2 anti-polycystin-2 antibody [7]. As reported, YCC2 detects a single band of ~110 kDa on western blots of protein extracted from mouse kidney and cultured human kidney cells (Figure 1A). Pretreating the antibody with recombinant polycystin-2 blocks this binding, Figure 1. Subcellular localization of polycystin-2 in cultured kidney cells. (A) Western blots of proteins extracted from mouse kidney and cultured human RPTEC cells. Blots were probed with a 1:5000 dilution of YCC2 anti-polycystin-2 sera. Bacterially expressed polycystin-2 antigen was added to the blot on the right as a control to show that the antibodies are recognizing polycystin-2 (PKD2). (B) Immunofluorescence images of human RPTEC cells labeled with antibodies to polyglutamylated ciliary tubulin (red, left panel) and polycystin-2 (PKD2) (green, right panel). Both are composite images made by combining one image taken in the plane of …